Using the Drosophila embryo as a model system, we have the exceptional opportunity to investigate how multiple cis-regulatory modules (CRMs) cooperate to support spatiotemporally-regulated gene expression during the course of development. In studies supported by the parent grant, we found that the Dorsal transcription factor, which is instrumental for patterning the dorsal-ventral (DV) axis, exhibits dynamics as do its target genes. Significant changes in levels of Dorsal and its target genes were observed both between as well as within each nuclear cycle, on the order of minutes. Chromatin immunoprecipitation experiments were also conducted to examine in vivo DNA occupancy by transcription factors, important for embryonic patterning. These experiments showed that many genes in the embryo are regulated by pairs of concurrently active cis-regulatory modules (CRMs) that drive expression in similar spatiotemporal patterns. The experiments proposed here aim to understand why and how multiple CRMs coordinate to control spatiotemporal gene expression in the Drosophila embryo, and, in particular, to provide insight into how transfer of activity from one CRM to the next is regulated to support developmental progression. We will capitalize on ample background information and our knowledge of DV patterning to help guide choice of particularly relevant cis-regulatory systems for study. Many molecular and genetic tools are available to support these studies in Drosophila, including ease of genetic approaches as well as the ability to manipulate large transgenes through recombineering, that facilitate functional assays of CRMs and other regulatory sequences in native context. Also, we will investigate the impact of chromatin conformation on gene expression using standard techniques (3C) as well as a novel imaging approach we are developing, to provide insight into when and how particular CRMs interact with the promoter with temporal and spatial resolution. Here in this renewal application we propose three specific aims that will significantly advance our understanding of how CRM coordinate to support developmental gene expression: Aim 1 - To analyze how transition from one CRM to the next supports continuous gene expression. Aim 2 - To investigate the relationship between gene expression dynamics, boundaries, and levels. Aim 3 - To assay chromatin conformation in vivo on a cell-by-cell basis. Carefully timed and spatially controlled expression of genes is required for normal development to proceed. As roughly a third of all CRMs controlling dorsal-ventral patterning may function as 'coordinate pairs', we argue that co-regulation of gene expression by integrative function of multiple CRMs is likely a general mechanism of cis-regulatory control; one that is just beginning to come to light. Splitting cis-regulatory information across multiple modules that jointly influence gene expression may provide flexibility of output that can be advantageous, especially for the developing embryo that is presented with genetic perturbation and/or changing environmental conditions. The conservation of gene regulatory mechanisms across all animals promises that these studies will have far reaching implications.